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Essential Requirement for IRF8 and SLC15A4 Implicates Plasmacytoid Dendritic Cells in the Pathogenesis of Lupus

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Essential Requirement for IRF8 and SLC15A4 Implicates Plasmacytoid Dendritic Cells in the Pathogenesis of Lupus Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus Roberto Baccalaa,1,2, Rosana Gonzalez-Quintiala,1, Amanda L. Blasiusb,1, Ivo Rimanna, Keiko Ozatoc, Dwight H. Konoa, Bruce Beutlerb,d,2,3, and Argyrios N. Theofilopoulosa,2,3 aDepartment of Immunology and Microbial Science and bDepartment of Genetics, The Scripps Research Institute, La Jolla, CA 92037; cProgram in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and dCenter for Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390 Contributed by Bruce Beutler, December 28, 2012 (sent for review December 21, 2012) In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) IFN-producing cells (22, 23). These cells, known as plasmacytoid are intimately involved in the pathogenesis of lupus. However, it DCs (pDCs), are the most potent producers of type I IFNs, a remains to be determined whether these cells are required in vivo functional characteristic attributed to constitutive expression of for disease development, and whether their contribution is re- TLR7, TLR9, and IRF7 and likely signaling from a unique in- stricted to hyperproduction of type I IFNs. To address these issues, tracellular compartment (24–27). The involvement of pDCs in lu- we created lupus-predisposed mice lacking the IFN regulatory factor pus is further suggested by the reduced frequency of these cells in 8 (IRF8) or carrying a mutation that impairs the peptide/histidine patient blood together with increases in afflicted organs, pre- fl transporter solute carrier family 15, member 4 (SLC15A4). IRF8- sumably caused by the attraction of activated pDCs to in ammatory fl deficient NZB mice, lacking pDCs, showed almost complete absence sites (10). Similar increases have been noted in in ammatory tissues ’ of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibod- of patients with Sjögren s syndrome (28), rheumatoid arthritis (29, ies, along with reduced kidney disease. These effects were observed 30), dermatomyositis (31), and psoriasis (32). despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 Collectively, these results suggest that pDCs, acting through stimuli and intact humoral responses to conventional T-dependent type I IFN hyperproduction, are major pathogenic contributors to lupus. Whether the participation of these cells is obligatory and -independent antigens. Moreover, Slc15a4 mutant C57BL/6- lpr remains to be documented in vivo, however. Here, using con- Fas mice, in which pDCs are present but unable to produce type I genic lupus-predisposed mice lacking pDCs (as well as other DC IFNs in response to endosomal TLR ligands, also showed an absence subsets) owing to IRF8 deficiency, or exhibiting pDC-specific of autoantibodies, reduced lymphadenopathy and splenomegaly, defects in endosomal TLR signaling and type I IFN production and extended survival. Taken together, our results demonstrate that owing to Slc15a4 (feeble) mutation, we provide strong evidence pDCs and the production of type I IFNs by these cells are critical that pDCs are indeed required for disease development, and this contributors to the pathogenesis of lupus-like autoimmunity in effect appears to be mediated by hyperproduction of inflamma- these models. Thus, IRF8 and SLC15A4 may provide important tar- tory cytokines, most likely type I IFNs. gets for therapeutic intervention in human lupus. Results + xtensive evidence suggests that type I IFNs are major patho- Absence of pDCs and CD8α DCs in IRF8-Deficient NZB Mice. Studies Egenic effectors in lupus-associated systemic autoimmunity. A in normal background mice have shown that IRF8, a hemato- well-documented pattern of expression of type I IFN-inducible poietic cell-specific transcription factor, is essential for the de- − − genes occurs in peripheral blood mononuclear cells of patients velopment of pDCs (33–35). Accordingly, assessment of Irf8 / with systemic lupus erythematosus (SLE) (1–3), and reduced dis- NZB mice at a young age (3 mo) showed almost complete ab- – + + + ease is observed in some lupus-predisposed mice that either lack sence of pDCs (CD11clowCD11b B220 Siglec-H PDCA1 )in the common receptor (IFNAR) for these cytokines (4, 5) or have spleen (Fig. 1A), and this correlated with undetectable in vivo been treated with IFNAR-blocking antibody (6). Consequently, production of type I IFNs in response to CpG-DNA (Fig. 1B). + attention has focused on defining the cell subsets and signaling Moreover, CD8α conventional DCs (cDCs) were absent and – – − − processes involved in type I IFN production, the mechanisms by CD4 CD8 cDCs were reduced in Irf8 / mice (Fig. 1C), con- which these mediators accelerate disease, and approaches to in- sistent with the levels of IRF8 expression in these subsets of terfere with these pathogenic events. normal mice (35). Total cDC numbers were unaffected, however, + Early in vitro studies showed that type I IFN production can be likely owing to compensatory increases in CD4 cDCs (Fig. induced in normal blood leukocytes by SLE autoantibodies com- 1C), which do not express IRF8 (35). As noted previously with − − plexed with nucleic acid-containing apoptotic/necrotic cell mate- Irf8 / normal background mice (36, 37), IRF8-deficient NZB + rial, and further work demonstrated that this activity is sensitive mice also showed expansion of marginal zone and CD21lowCD23 to RNase and DNase digestion (7, 8). These results were inte- follicular and transitional B cells, but total B-cell and T-cell num- grated in a more comprehensive scheme following the demon- bers were unchanged (data not shown). stration that type I IFN induction by these complexes is mediated by the engagement of endosomal Toll-like receptors (TLRs) (9– 11). Similarly, antigenic cargo containing nucleic acids was found Author contributions: R.B., K.O., D.H.K., B.B., and A.N.T. designed research; R.B., R.G.-Q., to promote B-cell proliferation in a TLR9- or TLR7-dependent A.L.B., and I.R. performed research; R.B., R.G.-Q., A.L.B., I.R., B.B., and A.N.T. analyzed manner, with this effect enhanced by type I IFN signaling (9, 12, data; and R.B. and A.N.T. wrote the paper. 13). The contribution of nucleic acid-sensing TLRs to systemic The authors declare no conflict of interest. autoimmunity was further corroborated by studies in lupus-pre- 1R.B., R.G.-Q., and A.L.B. contributed equally to this work. disposed mice lacking or overexpressing TLR7 and/or TLR9 (14- 2To whom correspondence may be addressed. E-mail: [email protected], Bruce.Beutler@ 20), and in Unc93b1 (3d) mutant mice in which signaling by UTSouthwestern.edu, or [email protected]. endosomal TLRs is extinguished (21). 3B.B. and A.N.T. contributed equally to this work. The cell population involved in type I IFN production in re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sponse to lupus-related immune complexes corresponds to natural 1073/pnas.1222798110/-/DCSupplemental. 2940–2945 | PNAS | February 19, 2013 | vol. 110 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1222798110 Downloaded by guest on September 30, 2021 Fig. 1. Absence of pDCs and CD8α+ cDCs in young IRF8-deficient NZB mice. Mutant and WT NZB mice (age 3 mo; n = 3–6/group) were analyzed for cellular differences in spleen and in vivo responses to CpG challenge. (A) pDC frequency and numbers. Spleen cells were assessed by flow cytometry using anti-CD11c and anti-PDCA-1 antibodies. Similar results were obtained when cells were stained with antibodies to B220, SiglecH, and CD11b (data not shown). (B) In vivo type I IFN production in response to TLR9 engagement. Serum IFN-α levels in CpG-challenged mice were determined by ELISA. (C) cDC frequency and numbers. + + + – – Spleen cells were analyzed by flow cytometry after gating on CD11c cells to identify CD4 , CD8 , and CD4 CD8 cDC subsets. Numbers within the flow cytometry plots correspond to average frequencies of the indicated subsets. Error bars in graphs indicate SD. Asterisks indicate statistical significance (P < 0.05). Reduced Autoimmunity in IRF8-Deficient NZB Mice. At age 11 mo, cDC subtypes present in these mice could not compensate for serum levels of polyclonal IgM and IgG, as well as IgG1 and the absence of pDCs in response to this TLR9 stimulus. Fur- − − + IgG2a, were similar in Irf8 / and WT NZB mice, whereas IgG2b thermore, CD11c cDCs derived from GM-CSF–differentiated was slightly increased and IgG3 was decreased in the mutant mice bone marrow (BM) cells showed reduced CD86 up-regulation (Fig. 2A). Strikingly, however, autoantibody production was al- and IL-6 production after in vitro stimulation with TLR7 or most completely suppressed in the IRF8-deficient mice. Thus, IgG TLR9 ligands in the presence or absence of IFN-α (Fig. 4). Thus, − − anti-erythrocyte autoantibodies, which are typically detectable in in addition to pDC deficiency, Irf8 / NZB mice exhibited de- − − 100% of WT mice by age 7–8 mo, were undetectable in Irf8 / fective cDC responses to endosomal TLR stimulation. mice even at 11 mo (Fig. 2B). Moreover, IgG2a anti-chromatin autoantibodies (the dominant subclass in WT NZB mice) and IgG B-Cell Responses to TLR Ligands and Exogenous Antigens Are Not anti-nuclear autoantibodies (ANA) were reduced to background Compromised in IRF8-Deficient NZB Mice. IRF8 is known to affect levels, and IgM anti-chromatin autoantibodies were decreased B-cell differentiation and germinal center formation (40), changes substantially (Fig. 2 C and D). Mirroring these serologic changes, that might contribute to reduced autoimmunity in IRF8-deficient − − kidney disease was significantly ameliorated in Irf8 / mice, as mice. To address this possibility, we assessed in vitro B-cell evidenced by reductions in both glomerulonephritis scores (Fig. responses to TLR or BCR engagement and in vivo antibody 2E) and immune (IgG2a, C3) deposits (Fig.
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